The use of short exogenous antisense oligonucleotides (ONs) or siRNA to silence gene expression on an RNA level has become an immensely popular approach to study fundamental functions of genes, to detect genes of interest, and to design new drugs against diseases of genetic origin. For development of high impact therapeutics with broad application, strategies that directly target the gene offer a powerful alternative to conventional therapies.
Progress in sequence-selective targeting of double stranded DNA (dsDNA) has been accomplished with minor groove binding polyamides or by DNA triple-helix-based approaches using modified oligonucleotides or helix-invading peptide nucleic acids (PNAs). However, the utility of these methods is limited by the sequence-dependent microstructure of the minor groove of DNA duplexes (polyamides), by target sequence restrictions (triplex-based approaches), or by the necessity of non-physiological salt concentrations (PNAs). An attractive alternative approach was introduced with pseudo-complementary DNA (pcDNA), i.e., DNA duplexes containing modified purine and pyrimidines that do not form stable base pairs with each other, while allowing hybridization to natural complementary DNA. pcDNA is able to strand invade blunt ended duplexes containing terminal mixed sequence target regions and this strategy has been extended into pseudo-complementary PNA (pcPNA), which has been used to target mixed sequence internal target regions of double stranded DNA. Unfortunately, the positively charged lysine residues commonly used to increase pcPNA solubility and binding affinity may lead to self-inhibitory effects of strand invasion at high probe concentrations. The requirement for low salt concentrations during pcPNA-mediated strand invasion of mixed sequence dsDNA is a limitation for all experiments in biological media and for numerous biotechnological applications. Development of alternative strategies for sequence selective recognition of dsDNA at physiologically relevant salt concentrations is therefore highly desirable.